Gas chromatographic mass spectrometric sequence determination of osteocalcin, a gamma-carboxyglutamic acid-containing protein from chicken bone.
The complete primary structure of osteocalcin, the gamma-carboxyglutamic acid (Gla)-containing calcium-binding protein isolated from chicken bone has been determined by gas chromatographic mass spectrometry. The method involves decarboxylation of the dry, intact protein in a low pressure atmosphere of DCl under conditions which quantitatively convert Gla into gamma, gamma-dideuteroglutamic acid residues without resulting in peptide bond cleavage. After partial enzymatic or acidic hydrolysis, the peptide mixtures are converted (without isolation of individual peptides) to the N-trifluorodideuteroethyl O-trimethylsilyl polyamino alcohols and analyzed by gas chromatographic mass spectrometry. Peptide fragments containing former Gla residues behave like Glu-containing peptides, but their mass spectra are shifted upward by 2 mass units/Gla residue. Chicken osteocalcin contains 50 amino acids, three of which are Gla. The structure is highly homologous to the sequence of the corresponding protein isolated from bovine bone, and the relative sequence locations of the Gla residues and the disulfide bridge are conserved.
D015055:1-Carboxyglutamic Acid / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001842:Bone and Bones / Q000032:analysis*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification; D002645:Chickens; D002942:Circular Dichroism; D019286:Endopeptidase K; D010450:Endopeptidases; D008401:Gas Chromatography-Mass Spectrometry; D015675:Osteocalcin; D010434:Pepsin A; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D013820:Thermolysin; D014357:Trypsin
D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D015675:Osteocalcin; D015055:1-Carboxyglutamic Acid; D010450:Endopeptidases; D014357:Trypsin; D019286:Endopeptidase K; D010434:Pepsin A; D013820:Thermolysin